The MALDI method is a technique for premixing a matrix substance, which easily absorbs a laser beam and is easily ionized, with a sample to be measured, and for ionizing the sample by irradiating this with the laser beam, to analyze the sample that is hard to absorb the laser beam or the sample such as protein, which is prone to suffer damage due to the laser beam. Generally, the matrix substance is added to the sample as a solution, and this matrix solution takes in the substance to be measured which is included in the sample. Then, it is dried and the solvent of the solution is vaporized, and crystal grains inclusive of the substance to be measured precipitate. When the laser beam is irradiated on this, the substance to be measured is ionized through the interaction of the substance to be measured, the matrix substance, and the laser beam. The MALDI method makes it possible to conduct an analysis minimizing break up of high molecular compound having large molecular weight. In addition to that, the MALDI method has high sensitivity suitable for micro amount analysis so that it is used in various fields such as life science in recent years.
The matrix substances for MALDI are appropriately selected in accordance with types, characteristics, and ion polarities of a substance to be measured, and representative substances include 1,4-bisbenzene, 1,8,9-trihydroxy anthracene, 2,4,6-trihydroxy acetophenone, 2,5-dihydroxybenzoic acid, 2-(4-hydroxy phenyl azo) benzoic acid, 2-aminobenzoic acid, 3-aminopyrazine-2-carboxylic acid, 3-hydroxypicolinic acid, 4-hydroxy-3-methoxycinnamic acid, trans-indoleacrylic acid, 2,6-dihydroxy acetophenone, 5-methoxysalicylic acid, 5-chlorosalicylic acid, 9-anthracenecarboxylic acid, indoleacetic acid, trans-3-dimethoxy-hydroxycinnamic acid, α-cyano-4-hydroxycinnamic acid, 1,4-diphenyl butadiene, 3,4-dihydroxycinnamic acid and 9-aminoacridine, and the like.
In recent years, attention has been paid to a mass spectroscopy imaging method of directly visualizing two-dimensional distribution of biomolecules or metabolites on a section of a living tissue by use of a MALDI mass spectrometer, and devices for this have been developed (see Non-Patent Literature 1, for example). In the mass spectroscopy imaging method, a two-dimensional image representing the intensity distribution of ions having a specific mass-to-charge ratio can be obtained on a sample such as a living tissue section. Accordingly, it can be used to detect the distribution of a specific substance in a pathological issue such as cancer, which facilitates figuring out the progress of disease or verifying the therapeutic effect of prescription. Thus, it is expected to be used for various applications in the fields of medicine, drug development, and life science. It is noted that, in Non-Patent Literature 1, the mass spectrometer is called as a microscopic mass spectrometer since a mass spectrometer that is capable of mass spectroscopy imaging is normally capable of microscopic observation, but, in the present specification, it is referred to as an imaging mass spectrometer so as to clarify that the device is aimed at conducting a mass spectroscopy imaging.
In the mass spectroscopy imaging method, high spatial resolution is required to obtain a mass spectroscopy imaging image to which the distribution of a target substance is accurately reflected. One of significant factors that determines the spatial resolution of the imaging mass spectrometer utilizing MALDI is the grain size of the matrix substance in the prepared sample and its uniformity. Conventionally used methods of adding matrix with regard to the mass spectroscopy imaging method include the method of injecting matrix solution in an array form to a sample by an ink jet method, and the method of blowing with a spray or the like and applying the matrix solution to the sample. However, these methods have difficulties in enhancing the spatial resolution of mass spectroscopy imaging because of the following reasons.
When the matrix solution is sprayed on the sample with a spray device, for example, the crystal grain takes in the substance to be measured from a broader area than a targeted area. As a result, the positional information of the substance to be measured on the sample is impaired, and the boundary line of the region where a certain substance exists becomes unclear. On the other hand, in the case of the method of injecting the matrix solution by the ink jet method to add the matrix solution to the sample, measuring positions (spots) to which the matrix solution is added are placed in an array form, and therefore positional relationship between the measuring positions is secured. However, the size of the measuring positions depends on the liquid amount of the matrix solution, and may have a diameter of tens to hundred micrometers on the sample due to the restriction of the injectable minimum liquid amount. This prevents the size of the measuring positions from being reduced greatly, which automatically determines the spatial resolution. It is noted that this problem has been pointed out in Patent Literature 1.
When 2,5-dihydroxybenzoic acid (DHB), which is often used as the matrix substance, or the like is sprayed with a spray device, the crystals are formed in needles, having various lengths. In the process of ionization, due to the variety of the size of the crystals, the positional information of the substance to be measured on the sample is impaired, which makes it difficult to enhance the spatial resolution.
In view of the problem described above, Patent Literature 1 proposes a sample preparation method of, instead of using conventional matrix substance, attaching minute particles to a sample, where every particle has a core made of a metallic oxide covered with polymer. Results of mass spectroscopy imaging of a cerebellar section of a rat by this method are shown in Patent Literature 1. However, in this sample preparation method, the preparation procedure is complicated, and an increase in cost is inevitable because inexpensive existing matrix substances cannot be used. Also, in the case of existing matrix substances, components suited to be ionized by every substance are known, and therefore an appropriate matrix substance can be selected in accordance with the substance to be measured. However, in the new sample preparation method described above, there is no established knowledge what component can be detected or what component cannot be detected in an analysis.
Non-Patent Literature 2 discloses a sample preparation method that achieves high spatial resolution by use of existing matrix substances. In this method, in order to conduct a mass spectroscopy imaging of protein, a matrix film layer is formed by a vacuum vapor deposition method on the surface of a slide glass on which a sample is attached, and subsequently, the slide glass is placed in an ambient including vaporized solvent such as methanol, which enhances re-crystallization of the matrix substance inclusive of the substance to be measured. The inventors of the instant application have verified by experiment that this sample preparation method is quite effective in improving the spatial resolution of the mass spectroscopy imaging.
However, according to the experiments by the inventors of the instant application, it is revealed that the sample preparation method disclosed in Non-Patent Literature 2 is difficult to improve detection sensitivity.